Methods of screening a subject for FHL1 mutations associated with novel X-linked muscular myopathies

ABSTRACT

Four and a Half LIM domains protein 1 (FHL-1) mutations at positions 128 or 224 that are associated with X-linked muscular myopathy, methods of screening subjects to identify those susceptible to muscular myopathy including muscular dystrophy and cardiomyopathy and kits.

This application is a 371 filing of International Patent ApplicationPCT/CA2008/001062 filed Jun. 4, 2008, which claims the benefit ofapplication No. 60/933,251 filed Jun. 4, 2007.

FIELD OF INVENTION

The present invention relates to gene mutations. FHL1 mutationsAssociated With a Novel X-Linked Muscular Myopathies.

BACKGROUND OF THE INVENTION

Muscular dystrophies (MD) are defined as a group of inherited muscledisorders characterized by the progressive degeneration and weakness ofvoluntary skeletal muscle (Davies and Nowak, 2006). The various forms ofMD vary widely with respect to age of onset, incidence, pattern ofinheritance, rate of progression, and distribution and severity ofmuscle weakness. Certain muscular dystrophies can involve cardiac andsmooth muscle tissue. MD most commonly exhibits an X-recessive mode oftransmission, and is usually caused by mutations in the DMD gene onXp21.2. Resulting in deficiencies in dystrophin protein, DMD mutationscause rapidly progressive weakness and wasting of the proximal musclesin the lower body. Duchenne MD (DMD), the most common neuromusculardisorder, is caused by frameshift mutations that result in the completeabsence of functional dystrophin, whereas the phenotypically less severeBecker's MD is associated with missense and inframe deletions thatresult in reduced levels of functional dystrophin or expression ofpartially functional protein (Davies and Nowak, 2006). This structuralprotein functions to link the actin cytoskeleton with muscle fibremembranes across the sarcolemma, providing structural support to themuscle cell (Ervasti, 2007). The absence of dystrophin compromises thecomplex across the muscle, leading to degeneration of muscle tissue.Affecting 1 in 4,000 live male births, DMD is correlated with onsetbefore age 6 and a typical life span of 20-25 years; in contrast,Becker's MD has onset in adolescence or adulthood with symptoms similarto but generally less severe than DMD. These include musclepseudohypertrophy, proximal muscle atrophy, and rarely, cardiomyopathyand/or mental deficits.

Emery-Dreifuss MD (EDMD) is another form of late onset X-recessive MDcaused by deficiencies in the emerin protein, encoded by the EMD gene onXq28 (Ellis, 2006). EDMD is phenotypically distinct from other X-linkedMDs in that there is humeroperoneal distribution of muscle wasting,absence of muscle pseudohypertrophy, and at very high frequency,cardiomyopathy.

There is a need in the art to identify Four and a Half LIM domainsprotein 1 (FHL-1) mutations, and the proteins encoded therefrom that areassociated with muscular myopathies including muscular dystrophy andcardiomyopathy. LIM domains, named after their initial discovery in theproteins Lin11, Isl-1 & Mec-3, are protein structural domains, composedof two contiguous zinc finger domains, separated by a two-amino acidresidue hydrophobic linker. Further there is a need in the art to beable to screen for such mutations to identify individuals that have orare at risk for developing muscular myopathies, including musculardystrophy and cardiomyopathy.

SUMMARY OF THE INVENTION

The present invention relates to gene mutations. More specifically, thepresent invention relates to gene mutations associated with muscularmyopathies.

According to the present invention there is provided a proteincomprising amino acids 1-230 of SEQ ID NO:1, a fragment thereof or asequence exhibiting at least 70% identity thereto and comprising theamino acid sequence VAKKCX₁GX₂X₃NPIT (SEQ ID NO:4) wherein X₂ is anyamino acid except C; and X₁ and X₃ are independently any amino acid.

Preferably X₁ is A or S and X₃ is K, N or Q.

Also provided is the protein as defined above, wherein X₂ is tryptophan.

The present invention also provides a protein as defined above, whereinthe protein is defined by SEQ ID NO:2 or SEQ ID NO:3.

Also provided by the present invention is a nucleic acid comprising asequence

a) encoding the protein as defined above or a fragment thereof;

b) that is the complement of a sequence encoding the protein as definedabove, or a fragment thereof;

c) that is capable of hybridizing to a nucleic acid encoding the proteinas defined above or fragment thereof under stringent hybridizationconditions; or

d) that exhibits greater than about 70% sequence identity with thenucleic acid defined in a) or b).

Also provided by the present invention is a nucleic acid as definedabove wherein the fragment comprises the amino acid sequence GWK.

Also provided is a nucleic acid as defined above wherein X₂ istryptophan.

Also contemplated is the nucleic acid as defined above wherein theprotein is defined by SEQ ID NO:2 or SEQ ID NO:3.

The present invention also provides a method of screening a subject foran X-linked muscular myopathy comprising,

a) obtaining a biological sample from the subject, and;

b) assaying the sample for a nucleic acid encoding the protein asdefined above or a fragment thereof comprising the amino acid sequenceVAKKCX₁GX₂X₃NPIT (SEQ ID NO:4) wherein X₂ is any amino acid except C;and X₁ and X₃ are independently any amino acid, or

c) assaying the sample for the protein as defined above or a fragmentthereof comprising the amino acid sequence VAKKCX₁GX₂X₃NPIT (SEQ IDNO:4) wherein X₂ is any amino acid except C; and X₁ and X₃ areindependently any amino acid.

Also provided is a method as defined above, wherein the muscularmyopathy is a skeletal muscle myopathy, or a cardiomyopathy, forexample, but not limited to muscular dystrophy.

Also provided is a method as defined above, wherein X₂ is tryptophan.

The invention also provides a method as defined above wherein theprotein is defined by SEQ ID NO:2 or SEQ ID NO:3.

Further provided is the method as defined above, wherein the subject isa human subject.

Also provided is a method as defined above, wherein the biologicalsample is a blood sample.

Also provided is a method as defined above wherein assaying comprisesPCR, probe hybridization or sequencing.

The present invention also provides a kit comprising

-   -   i) a protein or fragment thereof that is associated with        muscular myopathy as described herein,    -   ii) an antibody that selectively binds to a protein or fragment        thereof associated with muscular myopathy as described herein,        rather than a wild-type protein not associated with the muscular        myopathy,    -   iii) one or more nucleic acid primers to amplify a nucleotide        sequence encoding a protein or fragment thereof which comprises        a mutation associated with an X-linked muscular myopathy as        provided herein,    -   iv) one or more nucleic acid probes of between about 9 and 100        nucleotides that hybridizes to the nucleotide sequence encoding        a protein or fragment thereof which comprises a mutation        associated with an X-linked muscular myopathy as provided        herein,    -   v) one or more reagents including, but not limited to buffer(s),        dATP, dTTP, dCTP, dGTP, or DNA polymerase(s),    -   vi) instructions for assaying, diagnosing or determining the        risk of a subject to muscular myopathy,    -   vii) instructions for using any component or practicing any        method as described herein, or any combination thereof.

The present invention also provides a FHL-1 protein comprising anisoleucine insertion at position 128. In a preferred embodiment proteincomprises the human isoform a, b or c amino acid sequence or an aminoacid sequence which is at least 70% identical thereto.

The present invention also provides a nucleotide sequence encoding theFHL-1 protein as defined above.

Also provided by the present invention is an antibody that selectivelybinds the FHL-1 protein as described above but preferably not a wildtype FHL-1 protein.

The present invention also provides a method of screening a subject foran X-linked muscular myopathy comprising

a) obtaining a biological sample from the subject;

b) assaying the sample for a nucleic acid encoding a FHL-1 proteincomprising an isoleucine insertion at position 128, or

c) assaying the sample for the FHL-1 protein comprising an isoleucineinsertion at position 128,

wherein the presence of the nucleic acid or protein indicates that thesubject has or is at risk of developing a muscular myopathy.

Also provided by the present invention are kits comprising FHL-1 proteinhaving an isoleucine insertion at position 128, a nucleotide sequenceencoding a FHL-1 protein comprising an isoleucine insertion at position128, a probe that may be employed to identify nucleotide sequencesencoding an isoleucine at position 128, primers that can amplify suchsequences, antibodies that recognize the proteins as defined above butpreferably not wild-type FHL-1 proteins, instructions for screeningsubjects, one or more reagents that can be used to use one or componentsof the kit or any combination thereof. Other components as describedherein or as would be known in the art can also be included and thislist is not meant to be limiting in any manner.

This summary of the invention does not necessarily describe all featuresof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIGS. 1A-C show pedigrees of three families. FIG. 1A shows the pedigreeof the X-linked postural muscular myopathy family. Family members fromwhom DNA samples were obtained are indicated by arrows (

). FIG. 1B shows UK family 2 pedigree members exhibiting muscularmyopathies. FIG. 1C shows UK family 3 pedigree members exhibitingmuscular myopathies.

FIG. 2 shows atrophy of the postural back muscles as clinically assessedin a patient in the early stages of disease. Atrophy of the deltoideusmuscle. Gluteus maximus, biceps brachii, triceps brachii, and lower armsappear normal. Biceps femoris (hamstring muscles), adductor magnus(thighs), abductor pollicis brevis and adductor pollicis longus (hand)show signs of atrophy.

FIG. 3 shows muscle biopsy of the vastus lateralis muscle (A.) andanterior tibial muscle (B). Muscle histology revealed a moderatemyopathy with a moderate perimysial and limited endomysial fibrosis. Inall biopsies, some round, autophagic vacuoles predominant in type 2fibers were detectable. These vacuolar changes were most prominent inpatient B. Additionally, centrally placed myonuclei were increased andrarely single fiber necrosis and granular myofiber degeneration wereseen.

FIG. 4 shows muscle biopsy of the vastus lateralis muscle (A.) andanterior tibial muscle (B). Myosin ATPase staining at acidic pH 4.3/4.6reveals type I (dark) and type II (light) muscle fibre distribution inpatients in the early stages of disease. Variability of fiber size wasincreased in all specimens, with diameters ranging between 20 to 100 μm,and most prominent in type 2 fibers. In NADH and COX histochemistrycentrally negative core-like lesions were detected in both patients,without any further mitochondrial alterations.

FIG. 5 shows linkage analysis to the DMD locus using polymorphic STRintragenic markers STR-44, STR-45, STR-48, STR-49, and STR-50 revealeddifferent haplotypes in the affecteds, conclusively excluding the DMDlocus. Recombination of markers STR-44, STR-48, STR-49, and STR-50 isevident, as illustrated by haplotypes.

FIG. 6 shows an ideogrammatic representation of the X-linked myopathywith postural muscle atrophy (XMPMA) locus on the distal arm ofchromosome X, the electropherograms indicating the wild-type (SEQ ID NO:40) and mutation sequence (SEQ ID NO: 41) for the Austrian XMPMA family,and the secondary structure of FHL1, indicating the position of theresulting amino acid substitution, C224W, relative to structuralfeatures in the protein (SEQ ID NO: 39).

FIG. 7 shows amino acid and nucleotide sequences as described herein andthroughout as well as several wild-type protein sequences known in theart. e.g., mRNA sequence NM_(—)001449 (SEQ ID NO:7) and amino acidsequence NP_(—)001440 (SEQ ID NO:8) of Human FHL1 isoform a: mRNAsequence AF098518 (SEQ ID NO:9) and amino acid sequence AAC72390 (SEQ IDNO:10) of Human FHL1 isoform b; mRNA sequence AF220153 (SEQ ID NO:11)and amino acid sequence AAF32351 (SEQ ID NO:12) of Human FHL1 isoform c;mRNA sequence (SEQ ID NO:13) and amino acid sequence (SEQ ID NO:14) ofHuman FHL1 isoform AK09170; mRNA sequence (SEQ ID NO:15) and amino acidsequence (SEQ ID NO:16) of Human FHL1 isoform AX747139; mRNA sequence(SEQ ID NO:17) and amino acid sequence (SEQ ID NO:18) of Mouse FHL1isoform NM_(—)010211; mRNA sequence (SEQ ID NO:19) and amino acidsequence (SEQ ID NO:20) of Mouse FHL1 isoform AK158966.

FIG. 8 shows a comparative analysis of the 4^(th) LIM binding domain ofFHL1 across several species, i.e., Human (SEQ ID NO:21), Rhesus (SEQ IDNO:22), Mouse (SEQ ID NO:23), Opossum (SEQ ID NO:24), Chicken (SEQ IDNO:25), Xenopus (SEQ ID NO:26), Zebrafish (SEQ ID NO:27) and Tetraodon(SEQ ID NO:28).

DETAILED DESCRIPTION

The following description is of a preferred embodiment.

We have identified a large multigenerational Austrian family displayinga novel form of muscular myopathy with an X-recessive mode ofinheritance. Affected individuals develop specific atrophy of posturalmuscles, with histology showing gradual atrophy of type I muscle fibers.Known X-recessive MDs were excluded by immunocytochemical staining,marker analysis and gene sequencing. Marker analysis revealedsignificant linkage at Xq26-q27. Haplotype analysis based on 250K arraySNP chip data of five affected individuals along with three unaffectedfamily members confirmed this linkage region on the distal arm of theX-chromosome (Xq26-q27) and enabled us to narrow down the candidateinterval to 26 Mb encompassing approximately 850 consecutive SNPs.Sequencing of functional candidate genes led to the identification of amutation within the four-and-a-half LIM domain 1 gene (FHL1), whichputatively disrupts the 4th LIM domain. FHL1 on Xq27.2, is highlyexpressed specifically in type I muscle fibers. Thus, we havecharacterized a new form of myopathy, X-linked myopathy with posturalmuscle atrophy (XMPMA), and identified FHL1 as the causative gene. Otherfamily studies also confirm FHL1 as the causative gene in X-linkedmyopathies and cardiomopathies, as described herein.

Proteins and Amino Acids

According to an embodiment of the present invention there is provided aprotein comprising amino acids 1-230 of SEQ ID NO:1, a fragment thereofor an amino acid sequence exhibiting at least 70% identity thereto andcomprising the amino acid sequence VAKKCX₁GX₂X₃NPIT (SEQ ID NO:4)wherein X₂ is any amino acid except C; and X₁ and X₃ are independentlyany amino acid. Preferably X₁ is A or S and X₃ is K, N or Q. In apreferred embodiment X₂ is tryptophan, for example, but not limited toas defined by SEQ ID NO:2 or SEQ ID NO:3.

An amino acid sequence exhibiting at least 70% identity thereto isunderstood to include sequences that exhibit 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% identity, orany value therein between to SEQ ID NO:1 or a fragment thereof. Further,the protein may be defined as comprising a range of sequence identity asdefined by any two of the values listed or any values therein between.

Any method known in the art may be used for determining the degree ofidentity between polypeptide sequences. For example, but without wishingto be limiting, a sequence search method such as BLAST (Basic LocalAlignment Search Tool; (Altschul S F, Gish W, Miller W, Myers E W,Lipman D J (1990) J Mol Biol 215, 403 410) can be used according todefault parameters as described by Tatiana et al., FEMS Microbial Lett.174:247 250 (1999), or on the National Center for BiotechnologyInformation web page at ncbi.nlm.gov/BLAST/, for searching closelyrelated sequences. BLAST is widely used in routine sequence alignment;modified BLAST algorithms such as Gapped BLAST, which allows gaps(either insertions or deletions) to be introduced into alignments, orPSI-BLAST, a sensitive search for sequence homologs (Altschul et al.,Nucleic Acids Res. 25:3389 3402 (1997); or FASTA, which is available onthe world wide web at ExPASy (EMBL—European Bioinformatics Institute).Similar methods known in the art may be employed to compare DNA or RNAsequences to determine the degree of sequence identity.

In an embodiment of the present invention, which is not meant to beconsidered limiting there is provided a FHL1 protein comprising an aminoacid insertion. In a further embodiment, there is provided a FHL1protein comprising an isoleucine amino acid insertion. In still afurther embodiment, there is provided an a FHL1 protein comprising128InsI. Any isoform, for example, but not meant to be limiting toisoforms a, b or c may comprise this amino acid insertion. Nucleotidesequences encoding such proteins are also encompassed by the inventionas described below.

Nucleic Acids

Also contemplated by the present invention is a nucleic acid comprisinga sequence

-   -   a) encoding the protein as described above, or a fragment        thereof;    -   b) that is the complement of a sequence encoding the protein as        described above, or a fragment thereof;    -   c) that is capable of hybridizing to a nucleic acid encoding the        protein as described above or fragment thereof under stringent        hybridization conditions; or    -   d) that exhibits greater than about 70% sequence identity with        the nucleic acid described in a) or b).

Without wishing to be limiting, representative examples of nucleic acidsencoding the proteins as defined above are provided by SEQ ID NOs:5 and6 wherein X is not cytosine (c) or any other nucleotide that producescysteine when translated.

The nucleic acids described above include nucleic acids that may beemployed to produce proteins which are associated with X-linked muscularmyopathy, probes which may be used to identify or diagnose subjectscarrying a mutation which causes or predisposes the subject to muscularmyopathy, antisense or short inhibitory RNA that may be used to modulateproduction of protein from genes associated with muscular myopathy or acombination thereof. The proteins, fragments thereof or nucleic acids asdescribed above also may be used to produce antibodies that selectivelyrecognize the proteins as described above preferably over wild-typeproteins known in the art.

In a preferred embodiment of the nucleic acids as described above, X₂ istryptophan. In a further embodiment of the method, the protein isdefined by SEQ ID NO:2 or SEQ ID NO:3. In still a further embodiment,the protein is a human FHL1 protein comprising an isoleucine amino acidinsertion at position 128 (128InsI).

Stringent hybridization conditions may be, for example but not limitedto hybridization overnight (from about 16-20 hours) hybridization in4×SSC at 65° C., followed by washing in 0.1×SSC at 65° C. for an hour,or 2 washes in 0.1×SSC at 65° C. each for 20 or 30 minutes.Alternatively, an exemplary stringent hybridization condition could beovernight (16-20 hours) in 50% formamide, 4×SSC at 42° C., followed bywashing in 0.1×SSC at 65° C. for an hour, or 2 washes in 0.1×SSC at 65°C. each for 20 or 30 minutes, or overnight (16-20 hours); orhybridization in Church aqueous phosphate buffer (7% SDS; 0.5M NaPO₄buffer pH 7.2; 10 mM EDTA) at 65° C., with 2 washes either at 50° C. in0.1×SSC, 0.1% SDS for 20 or 30 minutes each, or 2 washes at 65° C. in2×SSC, 0.1% SDS for 20 or 30 minutes each for unique sequence regions.

The present invention is further directed to a nucleotide constructcomprising the nucleic acid as described above operatively linked to oneor more regulatory elements or regulatory regions. By “regulatoryelement” or “regulatory region”, it is meant a portion of nucleic acidtypically, but not always, upstream of a gene, and may be comprised ofeither DNA or RNA, or both DNA and RNA. Regulatory elements may includethose which are capable of mediating organ specificity, or controllingdevelopmental or temporal gene activation. Furthermore, “regulatoryelement” includes promoter elements, core promoter elements, elementsthat are inducible in response to an external stimulus, elements thatare activated constitutively, or elements that decrease or increasepromoter activity such as negative regulatory elements ortranscriptional enhancers, respectively. By a nucleotide sequenceexhibiting regulatory element activity it is meant that the nucleotidesequence when operatively linked with a coding sequence of interestfunctions as a promoter, a core promoter, a constitutive regulatoryelement, a negative element or silencer (i.e. elements that decreasepromoter activity), or a transcriptional or translational enhancer.

By “operatively linked” it is meant that the particular sequences, forexample a regulatory element and a coding region of interest, interacteither directly or indirectly to carry out an intended function, such asmediation or modulation of gene expression. The interaction ofoperatively linked sequences may, for example, be mediated by proteinsthat interact with the operatively linked sequences.

Regulatory elements as used herein, also includes elements that areactive following transcription initiation or transcription, for example,regulatory elements that modulate gene expression such as translationaland transcriptional enhancers, translational and transcriptionalrepressors, and mRNA stability or instability determinants. In thecontext of this disclosure, the term “regulatory element” also refers toa sequence of DNA, usually, but not always, upstream (5′) to the codingsequence of a structural gene, which includes sequences which controlthe expression of the coding region by providing the recognition for RNApolymerase and/or other factors required for transcription to start at aparticular site. An example of a regulatory element that provides forthe recognition for RNA polymerase or other transcriptional factors toensure initiation at a particular site is a promoter element. A promoterelement comprises a core promoter element, responsible for theinitiation of transcription, as well as other regulatory elements thatmodify gene expression. It is to be understood that nucleotidesequences, located within introns, or 3′ of the coding region sequencemay also contribute to the regulation of expression of a coding regionof interest. A regulatory element may also include those elementslocated downstream (3′) to the site of transcription initiation, orwithin transcribed regions, or both. In the context of the presentinvention a post-transcriptional regulatory element may include elementsthat are active following transcription initiation, for exampletranslational and transcriptional enhancers, translational andtranscriptional repressors, and mRNA stability determinants.

The regulatory elements, or fragments thereof, may be operativelyassociated (operatively linked) with heterologous regulatory elements orpromoters in order to modulate the activity of the heterologousregulatory element. Such modulation includes enhancing or repressingtranscriptional activity of the heterologous regulatory element,modulating post-transcriptional events, or both enhancing/repressingtranscriptional activity of the heterologous regulatory element andmodulating post-transcriptional events. For example, one or moreregulatory elements, or fragments thereof, may be operatively associatedwith constitutive, inducible, tissue specific promoters or fragmentthereof, or fragments of regulatory elements, for example, but notlimited to TATA or GC sequences may be operatively associated with theregulatory elements of the present invention, to modulate the activityof such promoters within plant, insect, fungi, bacterial, yeast, oranimal cells.

There are several types of regulatory elements, including those that aredevelopmentally regulated, inducible and constitutive. A regulatoryelement that is developmentally regulated, or controls the differentialexpression of a gene under its control, is activated within certainorgans or tissues of an organ at specific times during the developmentof that organ or tissue. However, some regulatory elements that aredevelopmentally regulated may preferentially be active within certainorgans or tissues at specific developmental stages, they may also beactive in a developmentally regulated manner, or at a basal level inother organs or tissues within a plant as well.

By “promoter” it is meant the nucleotide sequences at the 5′ end of acoding region, or fragment thereof that contain all the signalsessential for the initiation of transcription and for the regulation ofthe rate of transcription. There are generally two types of promoters,inducible and constitutive promoters.

An inducible promoter is a promoter that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer the DNAsequences or genes will not be transcribed. Typically the protein factorthat binds specifically to an inducible promoter to activatetranscription is present in an inactive form which is then directly orindirectly converted to the active form by the inducer. The inducer canbe a chemical agent such as a protein, metabolite, growth regulator, ora physiological stress imposed directly by heat, cold, or toxic elementsor indirectly through the action of a pathogen or disease agent such asa virus.

A constitutive promoter directs the expression of a gene throughout thevarious parts of an organism and/or continuously throughout developmentof an organism. Any suitable constitutive promoter may be used to drivethe expression of the proteins or fragments thereof as described herein.Examples of known constitutive promoters include but are not limited tothose associated with the CaMV 35S transcript. (Odell et al., 1985,Nature, 313: 810-812).

The term “constitutive” as used herein does not necessarily indicatethat a gene is expressed at the same level in all cell types, but thatthe gene is expressed in a wide range of cell types, although somevariation in abundance is often observed.

The gene construct of the present invention can further comprise a 3′untranslated region. A 3′ untranslated region refers to that portion ofa gene comprising a DNA segment that contains a polyadenylation signaland any other regulatory signals capable of effecting mRNA processing orgene expression. The polyadenylation signal is usually characterized byeffecting the addition of polyadenylic acid tracks to the 3 prime end ofthe mRNA precursor.

The gene construct of the present invention can also include furtherenhancers, either translation or transcription enhancers, as may berequired. These enhancer regions are well known to persons skilled inthe art, and can include the ATG initiation codon and adjacentsequences. The initiation codon must be in phase with the reading frameof the coding sequence to ensure translation of the entire sequence. Thetranslation control signals and initiation codons can be from a varietyof origins, both natural and synthetic. Translational initiation regionsmay be provided from the source of the transcriptional initiationregion, or from the structural gene. The sequence can also be derivedfrom the regulatory element selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA.

The present invention further includes vectors comprising the nucleicacids as described above. Suitable expression vectors for use with thenucleic acid sequences of the present invention include, but are notlimited to, plasmids, phagemids, viral particles and vectors, phage andthe like. For insect cells, baculovirus expression vectors are suitable.For plant cells, viral expression vectors (such as cauliflower mosaicvirus and tobacco mosaic virus) and plasmid expression vectors (such asthe Ti plasmid) are suitable. The entire expression vector, or a partthereof, can be integrated into the host cell genome.

Those skilled in the art will understand that a wide variety ofexpression systems can be used to produce the proteins or fragmentsthereof as defined herein. With respect to the in vitro production, theprecise host cell used is not critical to the invention. The proteins orfragments thereof can be produced in a prokaryotic host (e.g., E. colior B. subtilis) or in a eukaryotic host (e.g., Saccharomyces or Pichia;mammalian cells, such as COS, NIH 3T3, CHO, BHK, 293, or HeLa cells;insect cells; or plant cells). The methods of transformation ortransfection and the choice of expression vector will depend on the hostsystem selected and can be readily determined by one skilled in the art.Transformation and transfection methods are described, for example, inAusubel et al. (1994) Current Protocols in Molecular Biology, John Wiley& Sons, New York; and various expression vectors may be chosen fromthose provided, e.g., in Cloning Vectors: A Laboratory Manual (Pouwelset al., 1985, Supp. 1987) and by various commercial suppliers.

In addition, a host cell may be chosen which modulates the expression ofthe inserted sequences, or modifies/processes the gene product in aspecific, desired fashion. Such modifications (e.g., glycosylation) andprocessing (e.g., cleavage) of protein products may be important for theactivity of the protein. Different host cells have characteristic andspecific mechanisms for the post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen by one skilled in the art to ensure thecorrect modification and processing of the expressed cardiac stem cellproliferation protein.

Methods of Screening

The present invention also provides a method of screening a subject foran X-linked muscular myopathy comprising,

a) obtaining a biological sample from the subject, the biological samplecomprising DNA or RNA if the sample is assayed for nucleic acid, orFHL-1 protein if the sample is assayed for protein, and;

b) assaying the sample for a nucleic acid encoding the protein asdefined above or a fragment thereof comprising the amino acid sequenceVAKKCX₁GX₂X₃NPIT (SEQ ID NO:4) wherein X₂ is any amino acid except C;and X₁ and X₃ are independently any amino acid, or

c) assaying the sample for the protein as defined above or a fragmentthereof comprising the amino acid sequence VAKKCX₁GX₂X₃NPIT (SEQ IDNO:4) wherein X₂ is any amino acid except C; and X₁ and X₃ areindependently any amino acid.

The present invention also provides a method of screening a subject foran X-linked muscular myopathy comprising,

a) obtaining a biological sample from the subject, the biological samplecomprising DNA or RNA if the sample is assayed for nucleic acid, orFHL-1 protein if the sample is assayed for protein, and;

b) assaying the sample for a nucleic acid encoding a FHL-1 proteincomprising an isoleucine insertion at position 128 (128InsI), or

c) assaying the sample for the FHL-1 protein comprising an isoleucineinsertion at position 128 (128InsI).

The FHL protein may be identical or substantially identical to humanFHL-1 protein isoform a, b or c, as described herein or it may besubstantially identical meaning comprising at least 70% identity, morepreferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5% or 99.9% identity thereto.

Also provided is a method as defined above, wherein the muscularmyopathy is a skeletal muscle myopathy, for example, but not limited tomuscular dystrophy. Alternatively, but not wishing to be limiting, themuscular myopathy may be a cardiomyopathy. Cardiomyopathies arespecifically contemplated as the affected individuals studied hereinappear to exhibit symptoms of such and/or die of heart related disease.

In the embodiment described above, it is to be understood thatidentifying the target nucleic acid, protein or both in the biologicalsample obtained from the subject, may be employed to identify a subjecthaving or being at risk for developing a muscular myopathy, for example,but not limited to an X-linked muscular dystrophy or cardiomyopathy

By the terms “assaying the sample for a nucleic acid” it is meanttesting and/or characterizing the sample provided by the subject for anucleic acid that encodes a protein as defined above and is meant toinclude without limitation hybridization assays, nucleotide sequencing,nucleotide PCR including, but not limited to RT-PCR, etc or anycombination thereof.

In a preferred embodiment of the method of screening as defined above,X₂ is tryptophan. In a further embodiment, which is not meant to belimiting, the protein is defined by SEQ ID NO:2 or SEQ ID NO:3. Also,while the method of screening may be practiced on a variety of subjects,preferably, the subject is a human subject.

The sample obtained from the subject may comprise any tissue orbiological fluid sample from which DNA or RNA may be obtained. Forexample, but not wishing to be limiting, DNA may be obtained from blood,hair follicle cells, skin cells, cheek cells, tissue biopsy, or thelike. In a preferred embodiment, the sample is blood.

The present invention also contemplates screening methods which identifyand/or characterize the proteins as defined above within biologicalsamples from subjects. Such samples may or may not comprise DNA or RNA.For example, such screening methods may employ immunological methods,for example, but not limited to antibody binding assays such as ELISAsor the like, protein sequencing, electrophoretic separations to identifythe proteins as described above in a sample. As will be evident to aperson of skill in the art, the screening methods allow for thedifferentiation of the proteins as defined herein from wild typeproteins known in the art.

Kits

Also provided by the present invention is a kit comprising one or moreproteins or fragments thereof that is associated with muscular myopathy,for example, but not limited to, a muscular dystrophy or cardiomyopathyas described herein, an antibody that selectively binds to a protein orfragment thereof associated with muscular myopathy, dystrophy, orcardiomyopathy as described herein, rather than a wild-type protein notassociated with muscular myopathy, dystrophy, or cardiomyopathy, one ormore nucleic acid primers to amplify a nucleotide sequence encoding aprotein or fragment thereof which comprises a mutation associated withan X-linked muscular myopathy, dystrophy or cardiomyopathy as describedherein, one or more nucleic acid probes of between about 9 and 100nucleotides that hybridizes to the nucleotide sequence encoding aprotein or fragment thereof which comprises a mutation or insertionassociated with an X-linked muscular myopathy, dystrophy orcardiomyopathy as described herein, one or more reagents including, butnot limited to buffer(s), dATP, dTTP, dCTP, dGTP, DNA polymerase(s),instructions for assaying, diagnosing or determining the risk of asubject to a muscular myopathy, dystrophy, or cardiomyopathy,instructions for using any component or practicing any method asdescribed herein, or any combination thereof.

In a further embodiment, which is not meant to be considered limiting inany manner, there is provided a method of producing a non-human animalthat comprises the protein as defined herein and throughout, the methodcomprising,

transforming the non-human animal with a nucleotide construct thatencodes the protein as defined above, preferably in the absence of thewild type FHL-1 protein, more preferably in the absence of all isoformsof the FHL-1 protein. As human subjects exhibit hypertrophy of specificmuscles, the method as defined above may be employed in animals, forexample, in beef, horses, poultry, swine or any other non-human animalto produce animals that may exhibit increased muscle mass in variousbody areas.

The present invention will be further illustrated in the followingexamples.

EXAMPLES Example 1 Materials and Methods

Clinical Assessment

Probands are from a multigenerational Austrian family displayingclinical features suggesting MD, but with clinical differences frompreviously described muscular dystrophies (FIG. 1). We identified living6 patients (all males). Neurological examination was performed by aneurologist trained in neuromuscular disorders (S.Q.). First-degreerelatives were examined when possible. Serum creatine kinase (CK) levelswere measured in all affected individuals and their family members.

Myosin ATPase Staining

Standard histological protocols were employed to stain for myosin ATPaseat acidic pH 4.3/4.6 and assess the distribution of type I (slow twitch)and type II (fast twitch) muscle fibre types. Procedures were performedon adductor, biceps, deltoideus, erector, extensor, flexor, frontalis,gastrocnemius, gluteus, latissimus, pectoralis, peronaeus, rectus,sartorius, soleus, tibialis, triceps, vastus muscles, etc.

Muscle Immunocytochemistry

Standard immunocytochemistry protocols were utilized to perform stainingfor dystrophin, adhalin, merosin, dysferlin, caveolin, α-dystroglycan,emerin, lamin A/C, desmin, β-slow myosin heavy chain, spectrin, andα-sarcoglycan following muscle biopsies of patient 50. Monoclonalantibodies were obtained from Novocastra Laboratories Ltd. (VisionBioSystems, U.K.) for spectrin (NCL-SPEC1), dysferlin (NCL-Hamlet),emerin (NCL-Emerin), and α-sarcoglycan (NCL-α-SARC). AdditionalNovocastra antibodies were used for dystrophin staining, specific to thedystrophin rod-like domain (NCL-DYS1), C-terminus (NCL-DYS2), andN-terminus (NCL-DYS3). Monoclonal antibodies were employed for merosin(MAB 1922; Chemicon, Germany), caveolin (Caveolin3; TransductionLaboratories, BD Biosciences, Europe), α-dystroglycan (KlonVIA4-1;Upstate Biotechnology, Europe), lamin A/C (Mouse Hybridoma Supernatant),desmin (M0760, Klon D33; Dako, Europe), and myosin (805-502-L001, LotL02279, Klon A4.951; Alexis Biochemicals, Europe) staining procedures.

Exclusion of the DMD Locus

Genomic DNA was extracted from blood samples using standard procedures.DNA was amplified by PCR with conditions for thermal cycling adaptedfrom the protocol set out by ABI Prism® Linkage Mapping Set v2.5.Denaturation was performed at 95° C. for 15 min, followed by 10 cyclesof 94° C. for 15 min, 55° C. for 15 sec, 72° C. for 30 sec. This wasfollowed by 20 cycles of 89° C. for 15 sec, 55° C. for 15 sec, and 72°C. for 30 sec, with a final extension step of 72° C. for 10 min.Reaction mix consisted of 50 ng genomic DNA, 0.1 μmol of each primer,and HotStart Taq Master Mix (Qiagen, Europe) in a reaction mix of 10 μL.Linkage analysis to the DMD locus was performed using standardtechniques as will be described under ‘Linkage analysis.’ Fivepolymorphic STR microsatellite markers surrounding the DMD gene, STR-44(DXS1238; 180-210 bp), STR-45 (DXS1237; 160-185 bp), STR-48 (DXS997;105-120 bp), STR-49 (DXS1236; 230-260 bp), and STR-50 (DXS1235; 230-260bp), were selected for this purpose. Forward primers were labelled attheir 5′ ends with either 5-carboxyfluorescein (FAM) or NEDfluorochromes. STR-44 (forward primer: TCC AAC ATT GGA AAT CAC ATT TCA A(SEQ ID NO:29); reverse primer: TCA TCA CAA ATA GAT GTT TCA CAG (SEQ IDNO:30)), STR-45 (forward primer: GAG GCT ATA ATT CTT TAA CTT TGG C (SEQID NO:31); reverse primer: CTC TTT CCC TCT TTA TTC ATG TTA C (SEQ IDNO:32)), STR-48 (forward primer: GCT GGC TTT ATT TTA AGA GGA (SEQ IDNO:33); reverse primer: GGT TTT CAG TTT CCT GGG TA (SEQ ID NO:34)),STR-49 (forward primer: CGT TTA CCA GCT CAA AAT CTC AAC (SEQ ID NO:35);reverse primer: CAT ATG ATA CGA TTC GTG TTT TGC (SEQ ID NO:36)), andSTR-50 (forward primer: AAG GTT CCT CCA GTA ACA GAT TTG G (SEQ IDNO:37); reverse primer: TAT GCT ACA TAG TAT GTC CTC AGA C (SEQ IDNO:38)).

Genome-Wide SNP Analysis: Mapping of a New Locus to Xq26-q27

A genome-wide 250K NspI Affymetrix SNP microarray was performed on fiveaffected cases (individuals 20, 29, 50, 11, and 45) and three unaffectedrelatives at the Microarray Facility at The Centre for Applied Genomics(Toronto, Canada). Capable of genotyping on average 250,000 SNPs, thesingle nucleotide polymorphisms are separated by a median physicaldistance of 2.5 Kb and an average distance of 5.8 Kb between SNPs(Affymetrix, Calif., USA). The average heterozygosity of these SNPs is0.30, with approximately 85% of the human genome found within 10 Kb of aSNP. SNP microarray gene chip data was subsequently analyzed using dCHIPsoftware.

Linkage Analysis

Multipoint X-recessive nonparametric linkage was computed usingeasyLINKAGE plus v5.02. Allele frequencies were considered equal. One cMwas assumed to be equivalent to 1 Mb.

Sequencing and Mutation Analysis of Candidate Genes (MBNL3, VGLL1,FGF13)

The National Center for Biotechnology Information Entrez Genome MapViewer, Ensembl Human Genome Server and GenBank databases were employedto locate known genes, expressed-sequence tags and putative new genesthat map to Xq26-q27. Exon-intron boundaries of the candidate sequenceswere determined by BLAST searches against the human genome sequencedatabase at the National Center for Biotechnology Information. Intronicprimers (primer sequences available on request) were used to amplify allexons of the functional candidate genes by PCR. PCR products weresequenced using the BigDye® Terminator 3.1 Cycle Sequencing Kit(Perkin-Elmer, Applied Biosystems). Sequencing reactions were loaded onthe ABI Prism® 3100 DNA Analyzer (Perkin-Elmer, Applied Biosystems) andgenerated data was collected using the ABI® DATA COLLECTION version 1.1,and subsequently analyzed using the DNA SEQUENCING ANALYSIS version 3.6software. Sequencing and mutation analysis were performed at the Centrefor Addiction and Mental Health (Toronto, Canada).

Example 2 Identification and Characterization of a Novel X-LinkedMuscular Myopathy

This current study is the first to describe a family affected by a mildX-linked MD that specifically features atrophy that is limited mainly totype I muscle fibers in postural muscles. This large multigenerationalAustrian family originates from the Czech republic, and six livingaffected members have been ascertained and examined to date. Pedigreeanalysis (FIG. 1) shows an X-linked pattern of inheritance. Clinicalassessment in all six patients as well as two now-deceased patients fromthis family revealed a fairly uniform and characteristic phenotype (SeeTable 1). All subjects appeared to show an athletic stature (FIG. 2),however more detailed examination revealed an almost selective atrophyand wasting of postural muscles, while other muscles were hypertrophic.Predominantly weak and atrophic muscles include the soleus, peroneuslongus, tibialis anterior, vastus medialis, erector spinae, lower partof the latissimus dorsi, and abductor pollicis muscles. Additionally,all patients had significant contractures of the Achilles tendon andhamstrings, a short neck and also a mechanically limited range of neckflexion and extension. Tendon reflexes, sensory examination and mentalstatus were normal. In all affected individuals scoliosis, back pain,gait problems and elevated creatine kinase levels were noted. Thepseudo-athletic musculature is likely to be a compensatory response tothe atrophy of the postural muscles. Cases were asymptomatic until theage of 30, and in six deceased family members who had suffered from thedisease there was a wide range in age of death (45-72 years), typicallyfrom heart failure but of unknown mechanism. It appears that familymembers with more active lifestyles show less severe phenotypes andslower progression of disease.

Muscle biopsies from affected individuals revealed dystrophic changes inpostural muscles with variation in fiber sizes, degeneration of muscleendurance type I fibers, increased fatty and connective tissue, andmultinucleated sarcomeres (FIG. 3). Immunocytochemical staining ofbiopsied muscle tissue revealed no deficiencies of proteins associatedwith either autosomal or X-linked forms of MD, including dystrophin andemerin. This is consistent with the clinical and apparentepidemiological differences that distinguish and typify this new type ofMD. Myosin ATPase staining revealed a gradual atrophy of high-oxidative,low-glycolytic, endurance type I muscle fibers in postural muscles.While patients in the early stages of the disease show a relativelynormal distribution of type I and type II fibers, as the diseaseprogresses there are decreased numbers of type I fibers, which appearatrophied (FIG. 4). Non-postural muscles, including, among others, thegluteus medius, gluteus maximus, biceps brachii, triceps brachii, lowerarms, latissimus dorsi, and extensor muscles, appear normal with respectto muscle fiber distribution and function (Table 2).

Three different antibodies were used to detect distinct domains of thedystrophin protein. Staining was faint, but not significantly differentthan unaffected individuals, suggesting this family does not display avariant form of DMD or Becker's MD. Adhalin staining was performed,which excluded autosomal-recessive limb-girdle MD 2C (LGMD2C), LDMD2D,LGMD2E, and LGMD2F. Normal merosin staining excluded congenital MD.Staining for dysferlin and caveolin allowed for exclusion of LGMD2B andLGMD1C, respectively. LGMD1I was excluded following α-dystroglycanstaining. The likelihood of this postural MD representing a variant formof X-recessive EDMD was diminished following normal emerin staining.LGMD2D (Duchenne-like autosomal-recessive MD) and spinocerebellar ataxiatype 5 (SCA5) were excluded following α-sarcoglycan (LGMD2D) andspectrin (SCA5) staining. Normal staining for lamin A/C, desmin, andβ-slow myosin heavy chain excluded autosomal-dominant EDMD2 and LGMD1B(lamin A/C), desminopathies (desmin), and distal myopathy MPD1 (myosin),respectively. Myotonic dystrophy 2 (DM2) and proximal myotonic myopathy(PROMM) were also suggested as possible causative factors, but moleculargenetic analysis revealed no mutations.

Immunocytochemical data and pedigree analysis suggested that this familydisplays an unsevere myopathy with multinucleated sarcomeres and apattern of recessive X-chromosome inheritance. To exclude thepossibility that the phenotype in this family is a variant form of DMDor Becker's MD, we performed linkage analysis to the DMD locus usingfive selected polymorphic STR microsatellite markers surrounding the DMDgene; STR-44 (DXS 1238), STR-45 (DXS 1237), STR-48 (DXS997), STR-49(DXS1236), and STR-50 (DXS1235). Different haplotypes were revealed inthe affecteds across the DMD locus, excluding this locus as thecausative gene in this family. Recombination of the intragenic markersSTR-44, STR-48, STR-49 and STR-50 was evident (FIG. 5). Subsequentscreening for mutations in the DMD gene was conducted by sequencing cDNAproximal to the area spanned by the intragenic markers, which ruled outintragenic recombination. Genotypes for markers across the X-chromosomewere analyzed. Multipoint lod scores were found to be significant forthe Xq26-q27 region (lod>3), giving further confirmation for exclusionof the DMD locus. Multipoint lod scores revealed positive,non-significant results for areas surrounding the candidate intervalthat was later specified by SNP analysis (FIG. 5). A genome-wide SNPgenotype analysis was performed on the five affected individuals alongwith three unaffected family members at The Centre for Applied Genomics(Toronto, Canada). A ˜250K NspI Affymetrix SNP micorarray was used, andsubsequent analysis using dCHIP implicated a candidate region onXq26-q27, the candidate region encompasses approximately 850 consecutiveSNPs.

Three candidate genes from the Xq26-q27 critical region that encodestructural proteins expressed in muscle were screened. Themuscleblind-like protein 3 (MBNL3), vestigial-like 1 (VGLL1) genefibroblast growth factor 13 (FGF13) were all sequenced from genomic DNA,but no coding mutations were identified.

Sequencing of the coding and 5′UTR region of FHL1 (NM_(—)001449)resulted in a transversion at position 672 C to G leading to the aminoacid substitution C224W. This mutation co-segregated with disease statuswithin the family, all 6 affected subjects were hemizygous and allobligate carriers were heterozygous for the mutated allele. The mutationwas not detected in mixed Caucasian and Austrian control chromosomes.

FHL1 is a member of LIM-only proteins, containing four and a half LIMdomains with a common consensus sequenceC-X2-C-X16-21-H-X2-C-X2-C-X2-C-X17-C-X2-C. LIM only proteins arezinc-binding proteins that are known to be play a role in cell signalingand transcriptional regulation. So far, 5 FHL proteins have beenidentified: FHL1-5 are known to act as transcription regulators.

The C224W mutation replaces a highly conserved cysteine of the fourthLIM domain of FHL1 which is one of the four cysteines needed for thecentral binding of a zinc ion. Mutations of conserved cysteines that arepart involved in zinc binding have been shown to have a highlydeleterious effect on the tertiary structure of the protein (Taira etal, 1994). Furthermore, the C224W mutation also is located in the firstnuclear localization signal (NLS1) of the alternatively expressedisoform FHL1b (SLIMMER), which might lead to impaired FHL1b protein fromshuttle between the cytoplasm and the nucleus (Brown S et al; J BiolChem. 1999 Sep. 17; 274(38):27083-91

FHL1 has at least 3 different isoforms (a, b and c), each with differenttissue specificities. The C224W mutation affects FHL1 isoforms a (themost prevalent isoform) and b, but not isoform c. Hence, mutationswithin different regions of the gene may affect specific isoforms, withother isoforms unaffected, and thus may have different phenotypicconsequences. Furthermore, FHL1 has a number of protein binding partnersthat bind to different LIM domains within the protein, and thus amutation affecting the conformation of one LIM domain may have differentphenotypic consequences to a mutation affecting a different LIM domain.

In summary, we have identified the gene FHL1, and its encoded protein,as responsible for a new form of muscular myopathy, XMPMA. Thephenotypic features described in the Austrian family, in particular thespecific atrophy of postural muscles and pseudo athleticism, may bespecific for mutations within the SRF and MyBPC1 (muscle fiber type1-specific isoform) and ERK2 binding regions of FHL1. Mutationselsewhere in the gene may result in a much more heterogeneous myopathicphenotype. This has considerable implications for diagnostic evaluation,screening and genetic counseling for patients (also carriers) withmuscular or myotonic dystrophy of unknown genetic cause, in particularwhere the familial nature indicates X-linked inheritance and where theBecker's/Duchenne's MD and Emery-Dreifuss MD loci have been excluded,but also for sporadic cases. Additional information concerning thisexample may be obtained from Windpassinger et al., The American Journalof Human Genetics 82, 88-99, January 2008 which is herein incorporatedby reference.

Example 3 UK Pedigrees (Families 2 an 3) Exhibiting Muscular Myopathies

Four 4 male individuals in 3 consecutive generations presented withslowly progressive hip and arm weakness with onset in the 3rd-4thdecades. The index patient showed prominent shoulder girdle and armhypertrophy, with CK levels elevated to 1300 U/l. Respiratory failurewas reported in two patients who died in their 50s. The UK family 2pedigree is shown in FIG. 1B.

A third family, with a putative diagnosis of Becker muscular dystrophywas identified, where 6 females and 6 males, spread over 5 generations,were affected. The UK family 3 pedigree is shown in FIG. 1C. In malepatients, age at onset was in the late teens-3rd decade, and presentingclinical symptoms were progressive limb-girdle weakness with prominentscapular winging. Muscle hypertrophy was not a prominent feature, whileneck/cervical rigidity or weakness and Achilles tendon contractures werereported in three patients. CK levels were around 1500-2200 U/l. Twopatients were wheelchair bound from their 30s. Respiratory and heartfailure in the late 40s-50s were the causes of death in 2 patients.Female mutation carriers presented with a similar but milder clinicalpicture with onset in the 5th decade or later and CK levels onlyslightly elevated at 300 U/l. One female patient died at the age of 88years due to congestive heart failure. The index patient presented withfirst symptoms of hip flexor weakness (MRC 4) and elevated serum CKlevels of around 1300 U/l at the age of 35 years. At that time he wasplaying competitive football and showed a very athletic habitus. Musclehypertrophy was most prominent in his shoulder girdle and arm muscles.Neck flexion was compromised by spinal rigidity. His lung functionshowed a FVC of 4.6 l (90%) in a sitting position and dropped to 4.0 l(78%) in a lying position. There were no additional clinical signs orsymptoms of an underlying skeletal muscle or heart disease. Nerveconduction studies and an EMG were normal. A muscle biopsy from thevastus lateralis showed type I fibre atrophy, variation in fibre size,with some measuring up to 125 μm in diameter, and a few necrotic fibres.Immunohistochemical and Western blot analysis for proteins of thedystrophin glycoprotein complex, emerin, dysferlin, caveolin and calpainwere normal. Mutation analysis of the genes for dystrophin and emerindid not reveal any abnormalities. The maternal grandfather of the indexpatient started to experience difficulties with walking at 42 years ofage and used a wheelchair for the last years of his life. He died ofrespiratory failure at 52 with the label of Becker muscular dystrophy.Two nephews of the grandfather were also labeled with Becker musculardystrophy and experienced slowly progressive muscle weakness in legs andarms from their early 40ies. One of them died in his 50's of respiratoryfailure.

Data for the Index Patient, Family 2:

-   Age of onset: 35-   CK: 1342 U/L-   EMG: normal-   Muscle MRI: N.D.-   Athletic habitus in early stages: yes-   Muscle biopsy: myopathic-   Cardiac involvement: normal heart evaluation-   Neck and Achilles tendons: short (AT)

The mutation c.381_(—)382insATC (leading to p.Phe127_Thr128insIle) wasidentified in the index patients of both families and segregates withthe phenotype. The F127_T128InsI mutation occurs within the second LIMdomain, and thus is present in all three isoforms of FHL1, a, b and c.In conclusion, the data presented herein shows that the same FHL1mutation may give rise to heterogeneous phenotypes, with X-linkedrecessive or dominant inheritance.

Example 4 Study of Cardiomyopathies in the Austrian XMPMA Family

Patients with the clinical diagnosis of XMPMA and their immediaterelatives were invited to participate in a study for cardiovascularinvestigation of XMPMA. Standard 12 lead ECGs were recorded in therecumbent position. The echocardiographic studies were all performed byone operator using a GE Vivid 7 scanner. Measurements were madeaccording to the standards of the American Society of Echocardiographyand analyses were performed using the software programs of the scanner.The doppler variables measured were the peak aortic and LVOT velocities,and transmitral flow for assessing the diastoly. Strain and strain ratemeasurements were obtained by the non-Doppler 2D strain imagingtechnique as well as with TDI technique. Genomic DNA and serum profile(enzymes) were extracted from blood samples with standard procedures.Also used were: Magnet Resonance Imaging; Intracardiac catheter withbiopsy of the left ventricle; Treadmill testing; ECG Holter monitoring.

The most common abnormality was T-wave inversion in V4-V6 and other ST-Twave changes, partly signs of left ventricular hypertrophy. All affectedfamily members had pathological treadmill tests with ST wave changes andarrhythmia with extrasystoles (whereas Holter ECG has not been doneyet). Left ventricular hypertrophy with thickening confined to the apexas well as involvement of the right ventricle was present in allaffected family members. The left ventricle was normal in size withnormal systolic but impaired diastolic function. No abnormalities of themitral valve and its supporting structures were seen, and no LVOTgradient. All affected patients had a dilated left atrium and increasedleft atrial volume. Tissue velocities, strain rate and strain are alsoreduced. All affected male members had elevated levels of serumcreatinine kinase, CK-MB, LDH, NT-pro BNP, Trop T and liver enzymes.Without wishing to be limiting in any manner, important clinicalfindings included symptoms from Dyspnoe New York Heart association classII.

All citations are hereby incorporated by reference.

The present invention has been described with regard to one or moreembodiments. However, it will be apparent to persons skilled in the artthat a number of variations and modifications can be made withoutdeparting from the scope of the invention as defined in the claims.

REFERENCES

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TABLE 1 Clinical evaluations for members of the XMPMA family fromAustria, including electromyogram, NCV, muscle MRI, histologicalexamination of biopsied tissue, and involvement of heart, and of tendonsin neck and Achilles heel. Neck Athletic and Patients Age of NCV Musclehabitus Muscle Heart Achilles ID onset CK level EMG studies MRI at onsetbiopsie affection tendon SK060666 26 620 myopathic normal Nd yes nd ?FM240432 30 500-900  myopathic normal Nd yes myopathic Cardiomyopathyshort with arrhythmia AJ020657 32 620 normal Selective yes myopathicDialtativ cardiomyopathy short muscle hypertrophic atrophie, bent spineAA030554 32 400-1774 myopathic normal Selectiv yes myopathic Normalheart short muscle evaluation atrophy, bent spine AF061160 30 780myopathic normal — yes nd Unkown short MF250358 30 700 myopathic normalSelective yes myopathic Hypertrophic short muscle cardiomyopathy atrophybent spine MW211168 31 550 myopathic normal Nd unkown myopathicHypertrophic short cardiomyopathy BJ180830 30 800-1200 myopathic normal—nd yes myopathic Respiratory short failure

TABLE 2 Type I and type II muscle fibre distribution in several musclesin a patient in progressed stages of disease. Muscles represented inbold display significantly high portion of type I muscle fibres. Thereis a pronounced decrease in the proportion of type I muscle fibres inpostural muscles; adductor magnus, biceps femoris, deltoideus, peronaeuslongus, soleus, tibialis anterior, and vastus medialis muscles showedgradual atrophy of type I slow-twitch muscle fibres, whereas manymuscles with a high percentage of fiber type II show mild to pronouncedhypertrophy. Average muscle fiber composition Muscle Typ I Typ IIatrophic hypertrophic normal Abductor digiti minimi 51.8 48.2 X Abductorpollicis brevis 63.0 37.0 X Abductor hallucis X Adductor magnus(surface) 53.5 46.5 Adductor magnus (deep) 63.3 36.7 X Adductor pollicis80.4 19.6 ? Biceps brachii (surface) 42.3 57.7 X Biceps brachii (Deep)50.5 49.5 X Biceps fernoris 66.9 33.1 X Brachioradialis 39.8 60.2 XDeltoideus (Surface) 53.3 46.7 X Deltoideus (Deep) 61.0 39.0 X I dorsalinterosseus 57.4 42.6 X Erector spinae (Surface) 58.4 41.6 X Erectorspinae (Deep) 54.9 45.1 X Extensor digitorum 47.3 52.7 X Extensordigitorum brevis 45.3 54.7 X X Flexor digitorum brevis 44.5 55.5 XFlexor digitorum profundus 47.3 52.7 X Frontalis 64.1 35.9 ?Gastroenemius (lat. head. Surface) 43.5 56.5 X Gastroenemius (lat. head.Deep) 50.3 49.7 X Gastroenemius (medial head) 50.8 49.2 X Gluteus mediusX Gluteus maximus 52.4 47.6 X Iliopsoas 49.2 50.8 ? Iliocostalis XInterspinales cervicis X Infraspinatus 45.3 54.7 X Longus capitis XLongus colli X Longisimus dorsi X Latissimus dorsi 50.5 49.5 Xmultifidus X Orbicularis oculi 15.4 84.6 X Obliqus capitis X Pectoralismajor (clavic. head) 42.3 57.7 ? Pectoralis major (sternal head) 43.156.9 ? Peronaeus longus 62.5 37.5 X Psoas X Rectus abdominis 46.1 53.9 XRectus femoris (lat. head. Surface) 29.5 10.5 X Rectus femoris (lat.head. Deep) 42.0 58.0 X Rectus femoris (medial head) 42.8 57.2 XRhomboideus 44.6 55.4 X X Sartorius 49.6 50.4 Semimembranosus Xsemispinalis X Soleus (Surface) 86.4 13.6 X Soleus (Deep) 89.0 11.0 XSplenius X Sternocleidomastoideus 35.2 64.8 X X Supraspinatus 59.3 40.7X Temporalis 46.5 53.5 X Tibialis anterior (Surface) 73.4 26.6 XTibialis anterior (Deep) 72.7 27.3 X Trapezius 53.7 46.2 X X Transversusoccipitalis Triceps surae X Triceps (Surface) 32.5 67.5 X Triceps (Deep)32.7 67.3 X Vastus lateralis (Surface) 37.8 62.2 X Vastus lateralis(Deep) 46.9 53.1 X Vastus medialis (surface) 43.7 56.3 X Vastus medialis(Deep) 61.5 38.5 X JOHNSON et al. (1973).

What is claimed is:
 1. A method of identifying a subject having or atrisk of developing X-linked muscular myopathy which comprises: assayinga biological sample obtained from the subject to determine the presenceor absence of a mutation in a Four-and-a-Half LIM domain-1 (FHL-1)protein or a mutation in a nucleic acid encoding the FHL-1 protein,wherein a mutation in the FHL-1 protein or the nucleic acid encoding theFHL-1 protein results in disruption of one or more LIM domains of theFHL-1 protein, wherein the presence of a mutation in the nucleic acid orthe FHL-1 protein indicates that the subject has or is at risk ofdeveloping a X-linked muscular myopathy.
 2. The method of claim 1,wherein the sample is assayed to determine: the presence or absence of amutation in a nucleic acid encoding a FHL-1 protein comprising aminoacids 1-230 of SEQ ID NO: 1, or a fragment thereof, wherein the mutatedprotein comprises the amino acid sequence VAKKCX₁GX₂X₃NPIT (SEQ ID NO:4)wherein X₂ is any amino acid except C; and X₁ and X₃ are independentlyany amino acid, or the presence or absence of a mutated FHL-1 proteincomprising amino acids 1-230 of SEQ ID NO: 1, or a fragment thereof,wherein the mutated protein comprises the amino acid sequenceVAKKCX₁GX₂X₃NPIT (SEQ ID NO:4) wherein X₂ is any amino acid except C;and X₁ and X₃ are independently any amino acid.
 3. The method as definedin claim 2, wherein X₂ is tryptophan.
 4. The method as defined in claim2, wherein the FHL-1 protein is defined by SEQ ID NO:2 or SEQ ID NO:3.5. The method of claim 1, wherein the subject is a human subject.
 6. Themethod of claim 1, wherein the biological sample is a blood sample. 7.The method of claim 1, wherein assaying comprises PCR, probehybridization, immunohistochemistry, nucleotide sequencing or proteinsequencing.
 8. The method of claim 1, wherein the muscular myopathy is askeletal muscle myopathy or a cardiomyopathy.
 9. The method of claim 8,wherein the muscular myopathy is muscular dystrophy.
 10. The method ofclaim 1, wherein the sample is assayed to determine: the presence orabsence of a mutation in a nucleic acid encoding a FHL-1 protein,wherein the mutation comprises an isoleucine insertion at position 128,or the presence or absence of a mutation in a FHL-1 protein comprisingan isoleucine insertion at position
 128. 11. The method of claim 1,wherein a mutation in the nucleic acid encoding FHL-1 consists of amissense mutation or an insertion.
 12. The method of claim 1, wherein amutation disrupts LIM domain 4 of FHL-1.
 13. The method of claim 1,wherein a mutation disrupts LIM domain 2 of FHL-1.
 14. The method ofclaim 1, wherein the mutation in the nucleic acid encoding FHL-1 affectsisoform a, b and c.
 15. The method of claim 1, wherein the mutation inthe nucleic acid encoding FHL-1 affects isoform a and b.